Many time-of-flight mass spectrometers acquire separate time-of-flight spectra which contain the signals of only a few ions in each case in rapid succession and consequently produce individual spectra which are full of gaps. Thousands of these individual spectra, which are scanned at very high frequencies producing tens of thousands of spectra per second, are then immediately processed to form a sum spectrum for obtaining usable time-of-flight spectra with fairly well characterized signals for the ion species of different masses.
Mass spectra are calculated from these time-of-flight spectra. The purpose of this time-of-flight mass spectrometer is to determine the masses of the individual ion species as accurately as possible. Mass spectrometer developers are currently occupied with improving the mass accuracies which can be achieved from 30 ppm to 10 ppm or from 10 ppm to 5 ppm, depending on the spectrometer concerned, but the long-term aim is 3 ppm or even 2 ppm.
The term “ppm” (parts per million), which is used to describe the accuracy, is defined as the relative accuracy of the mass determination in millionth parts of the mass. The accuracy is established statistically and, under the tacit assumption that the measurement scatter conforms to a normal distribution, is characterized by the width parameter of the measurement value distribution, sigma. This width parameter is given by the distance between the point of inflection and the maximum of the Gaussian normal distribution curve. According to the definition, the following applies: if the mass determination is repeated many times over, then 68% of the values will lie within the double sigma interval stretching between the two sides (i.e. between the points of inflection), 95.7% will lie inside the quadruple sigma interval, 99.74% inside the sixfold sigma interval and 99.9936% inside the eightfold sigma interval of the normal distribution curve for the error scatter.
These types of mass spectrometers are used in particular in molecular biochemistry for determining the masses of the peptides produced by the tryptic digestion of a protein etc. By searching a protein database, the protein can be identified from the accurately determined masses of the peptides produced by the digestion, the quality of the identification depending on the accuracy of the mass determination. Knowledge of the accuracy is needed to set the mass tolerance for the search—if it is desired that none of the virtual digestion peptides of the database be lost during the search and ignored for the identification, four times the value of the accuracy achieved is entered (defined as the single sigma of the normal distribution), for example. For a mass-spectrometric accuracy of 10 ppm, therefore, a mass tolerance of 40 ppm is entered to include all the virtual digestion peptides for the identification with a certainty of 99.9936%. However, at the same time, other proteins with virtual digestion peptides which happen to have a similar mass may be found by the search, so the search is no longer unambiguous. Entering smaller mass tolerances can help, but again, digestion peptides may be excluded because the mass measurement is too inaccurate and therefore lead to a poor evaluation of the search. Consequently, the only way out is to use a mass spectrometer which can deliver a mass determination which is very accurate.
In another field of applications, the elementary composition of small molecules in the mass range up to several hundred atomic mass units has to be determined from the measured mass of the ions. Here, too, a very high accuracy is required.
TABLE 1Error distribution widths 2 × sigma as a functionof the mass and accuracyAccuracy [ppm]:301053Time of2 × sigma2 × sigma2 × sigma2 × sigmaMass [u]flight [μs][ns][ns][ns][ns]1007.070.1060.0350.0180.01120010.000.1500.0500.0250.01550015.810.2370.0790.0400.0241,00022.360.3350.1120.0560.0342,00031.620.4740.1580.0790.0475,00050.000.7500.2500.1250.075
The two-sided distribution widths 2×sigma of the errors in the time-of-flight determination which precedes the mass determination are shown in Table 1 for a time-of-flight mass spectrometer which needs a time-of-flight of 50 microseconds for ions of mass 5,000 unified atomic mass units. (The “unified atomic mass unit” is a non-coherent SI unit with the abbreviation “u”, which is a legally stipulated mass unit in Germany. The US abbreviation is “amu”). The distribution widths 2×sigma correspond to the distance between the two points of inflection of the Gaussian normal distribution and are expressed in nanoseconds. For an accuracy of 5 ppm, the (averaged) time-of-flight of the ions of a mass of 1,000 atomic mass units must be determined accurately to 56 picoseconds (plus/minus 28 picoseconds). (The times of flight of the ions must be determined with a relative accuracy which is double that required for the relative mass accuracy in each case, since the masses are proportional to the squares of the times of flight.) These figures are not dependent on the length of the flight path of the apparatus—a shorter flight path requires a lower acceleration voltage for the ions.
TABLE 2Mass peak widths as a function of mass and mass resolutionResolution:5,00010,00020,00040,000Time ofMass [u]flight [μs]Width [ns]Width [ns]Width [ns]Width [ns]1007.071.410.710.350.1820010.002.001.000.500.2550015.813.161.580.790.401,00022.364.472.241.120.562,00031.626.323.161.580.795,00050.0010.005.002.501.25
Table 2 shows the full widths of the ion signals (often referred to as ion peaks) at half maximum (FWHM), which are the maximum allowed for the stipulated mass resolutions. These peak widths are also expressed in nanoseconds.
The accuracy requirements discussed above can only be satisfied when good mass resolution is achieved. The mass resolution R is defined as the mass value m divided by the linear width Δm at half the signal height, where the linear width Δm has to be measured in the same mass units as the mass m (R=m/Δm). There is no strict relationship between the mass resolution and the resulting accuracy of the mass determination. However, it is true that a better resolution also results in a better mass accuracy for the same number of ions in any one ion peak. The ions which are available are combined in a narrower signal band, the signal is higher and the signal shows less noise in the vicinity of the signal peak.
As a very approximate rule of thumb, the position of the signal can be precisely fixed at approximately {fraction (1/20)} of its width. This means that a resolution of approximately R=20,000 must be aimed at in order to achieve an accuracy of 5 ppm for the mass calculation. However, this only applies to solitary lines. For the peaks of a group of isotopes, this only applies when the isotope lines of the ion signal are relatively well resolved, i.e. when the valleys between the maxima are really well defined and if only one line is used for the mass determination. If the peaks of a group of isotopes overlap, then the desired mass accuracy cannot be achieved.
Since organic ions of higher molar masses show a large number of isotopes (see FIG. 1), if the isotopes are resolved, a special method for mass determination as described in DE 198 03 309 (corresponding to U.S. Pat. No. 6,188,064) can be applied. This method produces increased mass accuracies. The method, designated here as the “SNAP” method for the sake of simplicity, consists of integrating the well-known actual isotope structure complete into the measured signal group for the mass determination instead of using the ion signals of the isotope peaks on their own. The mass accuracy increases with the number of available peak flanks, since these determine the quality of the integration. With eight well characterized flanks, the mass accuracy can be improved by a good factor of two, providing the mass calibration curve is able to provide this accuracy. By using this method, a mass accuracy of 5 ppm can already be achieved with a mass resolution of approximately 1,000. (However, we must not lose sight of the fact that the accuracy striven for is 3 ppm or even 2 ppm.)
From Table 2, it can be seen that the signal widths are very narrow when the mass resolution aimed at is in the region of 20,000. The signal widths (always measured as the full widths at half peak height) are 0.3 to approximately 2.5 nanoseconds for masses of 100 to 5,000 atomic mass units. Even for a resolution of R=10,000, signal widths ranging from 0.7 to 5 nanoseconds are necessary.
In this type of mass spectrometer, secondary electron multipliers are used to measure the ion currents. These are in the form of multi-channel plates with channels ranging from 3 to 25 micrometers diameter arranged slightly diagonally to the plate surface to prevent the ions from simply passing through. Two channel plates are normally connected one behind the other with the channel angles offset to increase the amplification of the electron currents. The degree of amplification can be set to between 105 and 107. In other words, one ion is able to produce 105 to 107 secondary electrons which are captured by an electrode connected downstream. The detectors are of complex design (such as that shown in FIG. 5) in order not to produce any signal distortion—but the specialist will be familiar with the arrangements, so no further discussion about these detectors is necessary here. When used with a post amplifier, the system can in principle be adjusted so that a single ion will produce a signal which stands out significantly above the electronic noise.
However, the avalanche-like secondary electron multiplication taking place in each of the channels on the plate also causes the electron-current signal to spread. A signal of 1.1 nanoseconds width is produced from the impingement of a single ion, and this only by using the best pairs of channel plates currently available commercially. The signal widths of cheaper channel plates range from 1.4 to 2 nanoseconds. Significant progress in the future is not expected, since development in this technology is essentially exhausted.
So-called transient recorders with scanning rates up to 4 MHz can be used for scanning the amplified ion current. It is of interest to note here that this technology is also largely at a mature stage of development. While for other electronic components and systems the processing speeds have doubled approximately every 1.5 to a maximum of 3 years, in the area of transient recorders, there has been no increase in the scanning rate for about the last six years, in spite of the sharp competition between some of the companies—and no significant change is expected during the next few years either.
If the electron current curve from the channel plates is digitized at a rate of 4 GHz point by point by using a device such as a transient recorder containing an analog-to-digital converter, then the minimum signal width obtained for each ion is 1.1 nanoseconds when using the best equipment, irrespective of the mass of the ion. If the signal profiles of several ions are added together or if several ions of the same mass arrive simultaneously, then the signal widths will be even larger, since focusing errors in the mass spectrometer, non-compensated effects from the initial energy distributions of the ions before pulsing out and other effects will play a part. These effects will also give rise to additional signal smearing of the order of a nanosecond, which also depends on the mass of the ions in most cases. In particular, it must be borne in mind that different penetration depths of the ions into the channels of the multi-channel plates give rise to different trigger times for the electron avalanches. With an effective flight path of one meter, a scattering of penetration depths of just 10 micrometers gives rise to a time-of-flight scatter of plus/minus 5 ppm and, consequently, a mass scatter of plus/minus 10 ppm. These values are halved by doubling the flight path—this effect on the signal width, by the way, is the only one which (for a given scatter of penetration depths) can be improved by increasing the length of the flight path alone. Since, according to experience, all these contributions to the signal width add up pythagorically (i.e. forming the root of the sum of the squares of the widths), signal widths less than 1.1 nanoseconds certainly cannot be achieved and signal widths less than 1.5 nanoseconds can only be obtained with the very best spectrometers and detectors; in most cases, therefore, the real signal widths range from 2 to 5 nanoseconds.
However, these values are significantly higher than the values which are necessary for the desired resolution of R=20,000 (or even R=10,000). According to the rule of thumb mentioned above, therefore, the desired mass accuracy of 5 ppm cannot be achieved—at any rate, not over the whole mass range. In conclusion, it is not possible to simply digitize the electron currents with a transient recorder and add up the individual spectra because the resulting peak signal widths are not good enough. In practice, therefore, other methods are also used which should be briefly described here along with the prior art of the time-of-flight mass spectrometers.
FIG. 5 is a schematic diagram of the principle of a time-of-flight mass spectrometer with orthogonal ion injection. A beam of ions with different initial energies and flight directions enters the ion-guide system (4) through an aperture (1) in a vacuum chamber (2). A damping gas enters the ion-guide system simultaneously. In the gas, the ions are decelerated by collision on entry. Since a pseudo-potential for the ions is present in the ion-guide system and is at its lowest at the axis (5), the ions collect at the axis (5). At the axis (5), the ions spread out toward the end of the ion-guide system (4). The gas from the ion-guide system is pumped away by the vacuum pump (6) on the vacuum chamber (2).
At the end of the ion-guide system (4), there is a drawing lens system (7) which is integrated into the wall (8) between the vacuum chamber (2) for the ion-guide system (4) and the vacuum chamber (9) for the time-of-flight mass spectrometer. In this case, the drawing lens system (7) is made up of five apertured diaphragms and draws the ions from the ion-guide system (4) to form an ion beam of low phase volume which is focused into the pulser (12). The ion beam is injected in the x-direction into a pulser. Once the pulser has just been filled with passing ions of the preferred mass for analysis, a short voltage pulse ejects a broad package of ions perpendicular to the previous flight direction, and forms a broad ion beam which is reflected by the reflector (13) and measured by the ion detector (14, 15) at high time resolution. In the ion detector, the ion signal, which is amplified in a secondary electron multiplier in the form of a double, multi-channel plate (14), is transmitted to the 50Ω cone (15) by capacitance. The amplified signal is passed to an analog preamplifier via a 50Ω cable. The 50Ω cone is used to terminate the cable at the input end in order to prevent any signal reflection. Since these electrical signals are only a few nanoseconds wide, it is vitally important to make sure that the quality of their transmission is extremely high in order to avoid any further distortion. The signals of the preamplifier are then passed to the digitizing system.
As described above, in time-of-flight mass spectrometers with orthogonal ion injection, sections from the ion beam are injected periodically by a pulser into the drift region of the mass spectrometer. At the same time, initial ion distributions in terms of space and velocity are compensated for as much as possible. The ions are usually generated by electrospray outside the vacuum system of the mass spectrometer. Pulse rates, and therefore spectral scanning rates, of 10 to 30 kHz are used. The data in the tables above are based on a mass spectrometer with a pulse frequency of 20 kHz, thus allowing a time of flight of 50 microseconds for the heaviest ions. According to the prior art, the individual ion pulses, each of which produces one spectrum, only contain very few ions (although work on improving this is being carried out). It is particularly rare to find two or more ions in the mass signal for an ion species of one mass; normally an ion signal of one mass is generated by a few ions coming from a much larger number of spectral scans. (However, it must be noted that significant improvements are expected in the ion sources. These will produce ion currents which will be too large for the scanning methods described below to cope with.)
Because of the small number of ions in each pulse, time-to-digital converters (TDC) are used in all commercially available instruments of this type. If the electron current which comes from the multi-channel plates and is detected by an electrode exceeds a certain threshold, then the event is recorded. This event is recorded purely as a time value without any associated intensity. One ion alone will trigger this event. The time-to-digital converter cannot recognize the difference between an event triggered by a single ion and an event triggered by many ions arriving simultaneously. The time values are then used in a histogram of the events. This histogram is made up of many separate time intervals of equal size. For each time interval there exists a counter for the events which take place within this time interval. The histogram is normally generated in a section of the computer memory where a memory cell is provided for counting the events for each time interval. For example, a memory cell may be available as a counter for every 250 picoseconds. A spectrum over a maximum duration of 50 microseconds would then take up 200,000 memory cells, each for a time interval of 250 picoseconds. The events associated with the time values are summed up in these memory cells to give a histogram-type presentation of the time-of-flight spectrum.
By using a TDC, therefore, the times of the ascending flanks of the electrical signals are retained whether the electrical signal has been generated by a single ion or a cluster of several ions of the same mass and therefore the same time of flight. The width of the electron-avalanche signal does not broaden the peak width. For this reason, higher resolutions can be achieved than by using an ADC. However, there are serious disadvantages in using TDCs.
The first disadvantage of using time-to-digital converters is the limited measurement dynamics. If the ion beam which is injected into the time-of-flight mass spectrometer becomes so intense that several ions of the same mass in a single pulse are accelerated more often into the drift region of the time-of-flight mass spectrometer, the information concerning the number of these ions is lost. Although this can be corrected by a statistical calculation of the frequency of the individual events, this method of correction soon fails as the intensity of the beam increases.
The second disadvantage associated with time-to-digital converters is the dead time of the counter after the event has taken place. It is easy to see that, after one event has been triggered, the next event cannot be measured until the electron current of the multiplier drops below the trigger threshold again. The detector is therefore blind for the time of the width of the signal. This dead time increases when a second or even a third event occurs within the time period represented by the signal width since the width of the signal continues to increase and the electron current no longer drops below the trigger threshold. The second or third ion is not necessarily of the same mass, but can certainly be an ion which is one or two atomic mass units larger and therefore belongs to another isotope line. This behavior can be somewhat improved artificially by not using an absolute threshold but a threshold of the rate of rise, i.e. a threshold of the first derivative. However, this again only helps to a limited extent.
If the dead time affects the neighboring isotopic signals, this behavior of time-to-digital converters leads to a distortion of the signal intensities. The distortion increases with the intensity of the ion beam, since an increasing number of neighboring events are suppressed. The behavior is illustrated in FIGS. 1 and 3 (with associated text). FIG. 1 shows the calculated theoretical isotope frequency of quintuply charged insulin (monoisotopic molecular weight=5735.65 u) showing a signal group between m/z=1147 and m/z=1149.5 u on the mass scale (m=mass, z=the number of elementary charges of the ion). FIG. 3, on the other hand, shows a measured spectrum with frequency distortions using a TDC. The ratio of Peak 5 to Peak 2 should be 2:1 but is actually 1:1 because of the effect of the dead time.
However, if a multi-channel analog-to-digital converter with a rapid adding unit, such as the “averaging transient recorders” which are on the market, is used for the spectra instead of the time-to-digital converter and if the ion currents reproduced by the multi-channel plates and the post amplifier are simply added then, although the resolution is reduced, the correct isotope pattern is obtained. If the resolution is sufficient for using the SNAP method (for example, in the high mass range), then satisfactory mass accuracies are obtained. However, the resolution is frequently not sufficient, as can be seen by the isotope group of the quintuply charged insulin in FIG. 2 (with associated text). In this case, a 2 MHz transient recorder was used. FIG. 2 is thoroughly typical, since large molecular ions which have been generated by electrospray ionization always have so many charges that they show the isotope group with the highest intensity in the range between m/z=1,000 and m/z=2,000. Particularly in this m/z range, therefore, it is desirable to produce the highest resolution.
However, the time-of-flight mass spectrometer with orthogonal injection of a continuous ion beam is not the only problem area where the resolution is reduced by the detector. A very similar problem exists with the time-of-flight mass spectrometer with pulsed ionization by matrix-assisted laser desorption and ionisation (MALDI). In this case, basically only transient recorders with ADCs are used because, in most cases, the ion signal of an ionization pulse can represent many ions of the same mass. Typically, 50 to 500 or, in a few instruments, even a few thousand spectra are added. Also, with these MALDI time-of-flight mass spectrometers the peak width for the ion signals of ions of the same mass is often limited by the width of the electron avalanche in the multi-channel plate.